SeMac does not Inhibit Opsonophagocytosis of S. equi by Horse PMN
A whole blood based phagocytosis assay [13, 14] was used to test whether SeMac inhibits opsonophagocytosis of S. equi by horse PMNs. The bacteria were labeled with FITC, treated with S. equi-specific horse convalescent serum in the absence or presence of SeMac, and incubated with horse blood. Percentage of PMNs with phagocytosed S. equi was determined by flow cytometry. As expected, percentage of PMNs with phagocytosed S. equi with the serum treatment was significantly higher than that for S. equi without serum treatment. But the inclusion of SeMac in the assay did not affect opsonophagocytosis of S. equi (Fig. 5A), suggesting that SeMac does not inhibit opsonophagocytosis of S. equi by horse PMNs.
The inability of SeMac to inhibit S. equi opsonophagocytosis by horse PMNs may be due to its inability to cleave the majority of horse IgG. If this is true, SeMac should inhibit opsonophagocytosis of GAS by human PMNs, since SeMac efficiently cleaves human IgG. To test this idea, the phagocytosis assay was repeated using GAS and human blood. SeMac indeed inhibits the opsonophagocytosis of GAS by human PMNs (Fig. 5B). These results suggest that IgG endopeptidase activity of SeMac is critical for inhibition of phagocytosis.